Arrays of biopolymers, such as arrays of peptides or polynucleotides (such as DNA or RNA), are known and are used, for example, as diagnostic or screening tools. Such arrays include regions (sometimes referenced as features or spots) of usually different sequence biopolymers arranged in a predetermined configuration on a substrate. The arrays, when exposed to a sample, will exhibit a pattern of binding which is indicative of the presence and/or concentration of one or more components of the sample, such as an antigen in the case of a peptide array or a polynucleotide of particular sequence in the case of a polynucleotide array. The binding pattern can be detected by reading the array, for example, by observing a fluorescence pattern on the array following exposure to a fluid sample in which all potential targets (for example, DNA) in the sample have been labeled with a suitable fluorescent label.
Methods of fabricating biopolymer arrays can be fabricated using light directed methods, in situ synthesis methods or deposition of the previously obtained biopolymers. In known light directed synthesis methods the aim is to form an array of oligonucleotides on a surface by removing a photoremovable group from a surface, coupling a monomer to the exposed region of the surface, and repeating the process. The in situ synthesis methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, as well as WO 98/41531 and the references cited therein for synthesizing polynucleotides (specifically, DNA). Such in situ synthesis methods can be basically regarded as iterating the sequence of depositing droplets of: (a) a protected monomer onto predetermined locations on a substrate to link with either a suitably activated substrate surface (or with a previously deposited deprotected monomer); (b) deprotecting the deposited monomer so that it can now react with a subsequently deposited protected monomer; and (c) depositing another protected monomer for linking. Different monomers may be deposited at different regions on the substrate during any one iteration so that the different regions of the completed array will have different desired biopolymer sequences. One or more intermediate further steps may be required in each iteration, such as oxidation and washing steps.
The “deposition method” basically involves depositing previously obtained biopolymers at predetermined locations on a substrate which are suitably activated such that the biopolymers can link thereto. The deposited biopolymers may, for example, be obtained from synthetic or biological sources. Biopolymers of different sequence may be deposited at different regions of the substrate to yield the completed array. Washing or other additional steps may also be used. Typical procedures known in the art for deposition of polynucleotides, particularly DNA such as whole oligomers or cDNA, are to load a small volume of DNA in solution in one or more drop dispensers such as the tip of a pin or in an open capillary and, touch the pin or capillary to the surface of the substrate. Such a procedure is described in U.S. Pat. No. 5,807,522. When the fluid touches the surface, some of the fluid is transferred. The pin or capillary must be washed prior to picking up the next type of DNA for spotting onto the array. This process is repeated for many different sequences and, eventually, the desired array is formed. Alternatively, the DNA can be loaded into a drop dispenser in the form of an inkjet head and fired onto the substrate. Such a technique has been described, for example, in PCT publications WO 95/25116 and WO 98/41531, and elsewhere. This method has the advantage of non-contact deposition. Still other methods include pipetting and positive displacement pumps such as the Biodot equipment (available from Bio-Dot Inc., Irvine Calif., USA).
In array fabrication, the quantities of DNA available for the array are usually very small and expensive. Sample quantities available for testing are usually also very small and it is therefore desirable to simultaneously test the same sample against a large number of different probes on an array. These conditions require use of arrays with large numbers of very small, closely spaced spots (features). For example, typical arrays may contain more than a thousand or ten thousand features in an area no larger than about 2 to 4 cm2. During array fabrication however, the resulting fabricated array may not have an actual layout which is precisely the same as a target layout. For example, some features actually deposited may have errors in position, dimensions, or presence (and may not be present at all due to intermittent errors in the drop deposition apparatus).
Thus, during reading of an array by a user following exposure to a sample, a determination should be made as to the exact shape of regions that represent actual features. Incorrectly designating regions as features or otherwise incorrectly calculating a total signal value for a feature in a determination of results from the exposed array, can read to serious errors in result interpretation. Determination of array features can be done manually by an operator viewing a display of a signal image acquired from a sample exposed array, and manually designating (with a mouse or other graphical computer pointer device) features based on a subjective evaluation of the boundaries of strong signal regions. The results for such manually designated feature regions can then be processed by known routines (such as by adding the signal values of each pixel within designated features of a digital signal image to obtain a total feature signal value). However, with thousands of features on each array, such a manual procedure is very slow, and does not lend itself to reproducible results. Therefore, computer implemented routines which automatically determine feature (or other region) boundaries are preferred particularly where many sample exposed arrays are to be read and analyzed in an efficient high-throughput manner. These routines and subsequent processing of features to obtain processed results for each (such as final signal values for each feature), are referred to as feature extraction.
However, the present invention recognizes that even sophisticated feature extraction routines will not be able to account for every possible error in designating feature shapes. For example, a routine which establishes a feature boundary based only on regions of highest signal strength, may fail to recognize a region where features have been smeared due to a scratch or other contact with the array. The present invention recognizes then that while automated feature extraction methods are needed for high-throughput reading and processing of array data, it would be desirable to have some convenient and rapid method by which an operator or some other means can review what shapes (for example, feature boundaries) were assigned. The present invention further recognizes that while this can be reproduced by running the same feature extraction program on the raw signal data acquired during array reading such a method is computationally intensive and therefore time consuming. Thus, the present invention realizes that it would be desirable, particularly where many arrays are subject to automated feature extraction, to have some method by which an individual or other means can readily review the shapes assigned to feature or other regions even after feature extraction.